Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells.

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR-108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC-alpha (11.6-fold), nPKC-delta (5.3-fold), and nPKC-epsilon (22.0-fold) but not aPKC-zeta. Northern blot analysis indicated a significant (2.3-fold) increase in the 10 kb transcript of cPKC-alpha, a slight (1.3-fold) increase in that of nPKC-epsilon but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC-alpha, PKC-delta, and PKC-epsilon and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR-108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme-selective increases in the protein levels of PKC isozymes of osteoblastic UMR-108 cells upon growth to confluence which may be regulated at the transcriptional or the post-transcriptional level. The results from UMR-108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999-1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

Effect of receptor-selective retinoids on growth and differentiation pathways in mouse melanoma cells.

Treatment of B16 mouse melanoma cells with all-trans-retinoic acid (ATRA) results in inhibition of cell proliferation and induction of differentiation. Accompanying these events is an induction of retinoic acid receptor beta (RARbeta) expression, an increase in protein kinase Calpha (PKCalpha) expression, and enhanced activator protein-1 (AP-1) transcriptional activity. These cells express nuclear RARalpha and RARgamma and nuclear retinoid X receptors (RXR) alpha and beta constitutively. We tested the ability of receptor-selective retinoids to induce the biochemical changes found in ATRA-treated melanoma cells and also tested their effectiveness in decreasing anchorage-dependent and -independent growth. The RXR-selective ligand (2E,4E)-6-(5,6,7,8-tetrahydro-3,5,5,8, 8-pentamethyl-2-naphthalenyl)-3,7-dimethyl-2,4,6-octatrienoic acid (SR11246) was most effective at inhibiting anchorage-dependent growth, whereas the RARgamma-selective ligand 6-[(5,6,7, 8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)(hydroxyimino)methyl]-2-naphthalen ecarbo xylic acid (SR11254) was most potent at inhibiting anchorage-independent growth. In contrast, 4-(5,6,7,8-tetrahydro-5,5, 8,8-tetramethyl-2-naphthalenecarboxamido)-benzoic acid (Am580), an RARalpha-selective ligand, was the most effective receptor-selective agonist for inducing RARbeta mRNA and increasing the amount of PKCalpha protein. All of the retinoids induced a concentration-dependent increase in AP-1 transcriptional activity, with little difference in effectiveness among the receptor-selective retinoids. A synergistic increase in the amount of PKCalpha was found when an RAR-selective agonist was combined with an RXR-selective agonist. One possible explanation for this result is that an RXR-RAR heterodimer in which both receptors are liganded is required for maximum expression of this critical component of the ATRA-induced differentiation pathway. Our data suggest that synthetic retinoids can activate different growth and differentiation pathways preferentially in B16 melanoma cells, due, most likely, to their ability to activate a different subset of receptors.

Recent advances in the use of vitamin A (retinoids) in the prevention and treatment of cancer.

Vitamin A, its physiologic metabolites, and synthetic derivatives (retinoids) have been shown to have protective effects against the development of certain types of cancer. In addition, pharmacologic amounts of retinoids have been used with some success in the treatment of a few human tumors. The chemoprevention effect of retinoids is most likely exerted at the tumor-promotion phase of carcinogenesis. Retinoids block tumor promotion by inhibiting proliferation, inducing apoptosis, inducing differentiation, or a combination of these actions. Clinically, isotretinoin (13-cis-retinoic acid) significantly decreases the incidence of second primary tumors in patients with head-and-neck cancer and reduces appearance of non-melanoma skin cancer in patients with xeroderma pigmentosum. Retinoic acid has proved to be an effective treatment for promyelocytic leukemia. However, retinoid resistance limits its use as a single agent. Clinical trials are in progress to determine the efficacy of retinoids in treating other types of cancer such as neuroblastoma and breast carcinoma. The development of receptor-selective retinoids and selective inhibitors of retinoid metabolism may lead to further use of retinoids in both chemoprevention and treatment of cancer.

Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element.

Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC alpha mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC alpha gene. A 13 kb mouse genomic fragment containing the 5' flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at -93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC alpha expressed in these cell lines. Reporter gene assays showed that the region between -179 and -452 bp likely contains a silencer element(s). The promoter activity of a -179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (-93 to -65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC alpha gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.

Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro.

The mechanism by which retinoids, thyroid hormone (T3) and estrogens modulate the growth of breast cancer cells is unclear. Since nuclear type II nuclear receptors, including retinoic acid receptor (RAR), retinoid X receptor (RXR) and thyroid hormone receptor (TR), bind direct repeats (DR) of the estrogen response elements (ERE) half-site (5'-AGGTCA-3'), we examined the ability of estrogen receptor (ER) versus type II nuclear receptors, i.e. RARalpha, beta and gamma, RXRbeta, TRalpha and TRbeta, to bind various EREs in vitro . ER bound a consensus ERE, containing a perfectly palindromic 17 bp inverted repeat (IR), as a homodimer. In contrast, ER did not bind to a single ERE half-site. Likewise, ER did not bind two tandem (38 bp apart) half-sites, but low ER binding was detected to three tandem copies of the same half-site. RARalpha,beta or gamma bound both ERE and half-site constructs as a homodimer. RXRbeta did not bind full or half-site EREs, nor did RXRbeta enhance RARalpha binding to a full ERE. However, RARalpha and RXRbeta bound a half-site ERE cooperatively forming a dimeric complex. The RARalpha-RXRbeta heterodimer bound the Xenopus vitellogenin B1 estrogen responsive unit, with two non-consensus EREs, with higher affinity than one or two copies of the full or half-site ERE. Both TRalpha and TRbeta bound the full and the half-site ERE as monomers and homodimers and cooperatively as heterodimers with RXRbeta. We suggest that the cellular concentrations of nuclear receptors and their ligands, and the nature of the ERE or half-site sequence and those of its flanking sequences determine the occupation of EREs in estrogen-regulated genes in vivo .

Characterization of retinoic acid-induced AP-1 activity in B16 mouse melanoma cells.

Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells, which is accompanied by an increase in protein kinase Calpha (PKCalpha) as well as a selective enrichment of nuclear PKCalpha. We report here that RA also increases AP-1 activity in these cells. Transient transfection of B16 cells with luciferase reporter gene constructs indicated that RA induced a concentration-dependent increase in AP-1 activity. Acute treatment (2 h) of B16 cells with phorbol dibutyrate (PDB) increased AP-1 activity by 10-fold. RA treatment did not change the expression of Jun family members; however, it decreased the expression of c-Fos. In contrast acute PDB treatment induced c-Fos expression, while having little effect on c-Jun. Five DNA-protein complexes were formed with nuclear extracts from B16 cells and an oligonucleotide containing an AP-1 consensus sequence. Several complexes were decreased in cells treated with RA. Conversely, certain complexes were increased in cells acutely treated with PDB. The slowest migrating complexes were shown to contain Fos family members. Down-regulation of PKC inhibited both the acute PDB-induced and the RA-induced increase in AP-1 activity. The selective PKC enzyme inhibitor, bisindolylmaleimide, reduced PDB-stimulated AP-1 activity, but enhanced RA-induced AP-1 activity. These results together with our previous studies suggest the intriguing possibility that PKC protein, but not enzyme activity, may be required for RA-induced AP-1 activity.

Characterization of density-dependent regulation of the tyrosinase gene promoter: role of protein kinase C.

The rate-limiting step in melanogenesis is catalyzed by tyrosinase, a multifunctional enzyme encoded by the albino locus. We have previously reported that depletion of protein kinase C by long-term treatment of B16 mouse melanoma cells with phorbol dibutyrate (PDBu) prevented cell density-dependent melanogenesis. This was accompanied by a lack of induction of tyrosinase protein and mRNA. We report here the effect of PDBu on the functional activity of the mouse tyrosinase promoter by reporter gene assay and its effect on the binding of nuclear proteins from B16 cells to the "M-box" region of the mouse tyrosinase promoter. Short-term PDBu treatment of B16 cells transfected with a mouse tyrosinase promoter-luciferase construct resulted in increased reporter gene activity, while long-term PDBu treatment inhibited reporter gene activity. Using an oligonucleotide containing the M-box and its flanking residues in electrophoretic mobility shift assays, we found a density-dependent change in the pattern of DNA-protein complexes. One complex was found to be negatively regulated by long-term PDBu treatment. Competition experiments with various mutated oligonucleotides demonstrated that both the M-box and flanking residues are important for nuclear protein binding. The complex whose formation was inhibited by long-term PDBu treatment was shown to contain the basic helix-loop-helix leucine zipper protein microphthalmia-associated transcription factor (MITF). These results suggest that chronic PDBu treatment might inhibit tyrosinase expression (and subsequent melanogenesis) by affecting the amount or function of MITF.

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C.

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKC alpha had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKC alpha in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1.

Control of retinoic acid receptor expression in mouse melanoma cells by cyclic AMP.

Retinoic acid receptor (RAR) alpha and gamma mRNAs were constitutively expressed in B16 melanoma cells with or without retinoic acid (RA) treatment. RAR beta mRNA, however, was significantly expressed only after exposure to RA. Induction of RAR beta by RA occurred within 1 h and was not inhibited by cycloheximide (i.e., did not require new protein synthesis). All three RAR mRNA levels were dramatically decreased with 8-bromo-cyclic AMP treatment and could not be rescued by addition of RA. Analysis of RAR gamma revealed that this decrease occurred within 1 h of exposure to 8-bromo-cyclic AMP and was not blocked by simultaneous treatment with cycloheximide. The stability of RAR gamma mRNA was not altered by cyclic AMP treatment. Nuclear extracts from 8-bromo-cyclic AMP-treated cells showed a large decrease in protein binding to a retinoic acid response element (RARE) oligonucleotide compared to control cells. This correlated with a marked reduction of RA-stimulated RARE-reporter gene activity in transfected cells which were treated with cyclic AMP. Pretreatment of B16 cells with cyclic AMP prior to RA addition dramatically reduced induction of PKC alpha, an early marker of RA-induced cell differentiation. Thus, cyclic AMP can antagonize the action of RA most likely via its ability to inhibit RAR expression.

Inhibition of retinoic acid receptor function and retinoic acid-regulated gene expression in mouse melanoma cells by calreticulin. A potential pathway for cyclic AMP regulation of retinoid action.

Calcium is a second messenger that controls a wide variety of cellular functions. Because of its multiple actions, there is a stringent requirement for calcium homeostasis, and this is achieved in part by a system of transport and storage proteins such as calreticulin located in the endoplasmic reticulum. Calreticulin is also found in the nucleus, suggesting that it may have a role in transcriptional regulation. It has been reported that calreticulin can inhibit steroid-regulated gene transcription by preventing receptor binding to DNA. Here we report that overexpression of the calreticulin gene in B16 mouse melanoma cells resulted in a decrease in retinoic acid (RA)-stimulated reporter gene expression. Gel shift analysis showed that purified calreticulin inhibited the binding of endogenous RAR to a beta-RA response element oligonucleotide, only if added prior to the addition of the oligonucleotide. Co-immunoprecipitation studies suggest a physical interaction between RAR and calreticulin. Transfection of the calreticulin gene into B16 cells inhibited the RA induction of protein kinase Calpha, a marker of RA-induced differentiation. We also found that cyclic AMP increased the expression of calreticulin. Cyclic AMP may act to antagonize RA action by both decreasing RAR expression (Y. Xiao, D. Desai, T. Quick, and R. M. Niles, J. Cell Physiol., in press) and stimulating calreticulin levels.

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-BL6 melanoma variants.

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

The relationship between susceptibility to retinoic acid treatment and protein kinase C alpha expression in murine melanoma cell lines.

Retinoic acid (RA)-induced differentiation of B16 mouse melanoma cells is accompanied by a large increase in the amount of PKCalpha protein. Overexpression of PKCalpha in these cells results in a more differentiated phenotype. To determine if these findings had general applicability to murine melanomas, we investigated the relationship between sensitivity to RA and induction of PKCalpha in three different murine melanoma cell lines. RA inhibited the anchorage-dependent growth of all three cell lines, with JB/MS being the most sensitive, S91 intermediate, and RPMI the least affected. RA also inhibited soft agar colony formation in JB/MS, but had little effect on RPMI. All cell lines expressed PKCalpha, but not beta or gamma. RA induced a large concentration-dependent increase in PKCalpha protein in JB/MS (6- to 10-fold), a smaller increase in S91 (2- to 3-fold), and very little induction of PKCalpha in RPMI. Previously we had observed that the amount of PKCalpha increased with the density of B16 cells in culture. We found that this density-dependent increase in PKCalpha occurred in three out of four melanoma cell lines examined. These results suggest that PKCalpha plays an important role in RA-induced murine melanoma cell differentiation.

Retinoic acid specifically increases nuclear PKC alpha and stimulates AP-1 transcriptional activity in B16 mouse melanoma cells.

B16 melanoma cells differentiate upon treatment with retinoic acid (RA). This differentiation process is accompanied by an increase of protein kinase C alpha (PKC alpha) mRNA and protein levels. Overexpression of PKC alpha in these cells results in a more differentiated phenotype, suggesting the importance of this protein in the control of differentiation by RA. The purpose of the study reported here was to determine the subcellular distribution of the RA-induced PKC alpha, whether the RA-induced increase in PKC alpha protein levels was accompanied by an increase in in situ enzyme activity, and whether RA altered AP-1 transcriptional activity. We found that RA treatment increased PKC alpha protein levels in all subcellular compartments examined, but it also induced a selective enrichment in nuclear-associated PKC alpha levels. Treating cells with an active phorbol ester induced translocation of PKC alpha to membrane fractions, but had no effect on nuclear PKC alpha levels. RA also increased PKC enzymatic activity in intact cells as determined by phosphorylation of the PKC-specific endogenous substrate MARCKS. However, while RA induced a five- to eightfold increase in total cellular PKC alpha protein levels, it only increased MARCKS phosphorylation by twofold. In light of the increase in in situ PKC enzyme activity and the enrichment of nuclear PKC alpha, we determined whether AP-1 activity might be increased in RA-treated cells. Use of luciferase reporter gene constructs with or without AP-1 elements transfected into B16 cells indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity. These results suggest a hypothesis whereby RA-induced nuclear PKC alpha might lead to increased AP-1 activity and show that RA-induced growth inhibition and differentiation are not always accompanied by an inhibition of AP-1 activity as has been proposed by other investigators.

Expression and regulation of retinoid X receptors in B16 melanoma cells.

Recently, a new subfamily of nuclear retinoid receptors that is distinct from that of RARs has been identified and named Retinoid X receptors (RXRs). These receptors specifically bind 9-cis-retinoic acid (9cisRA), but not all-trans-retinoic acid (ATRA). We determined which RXR subtypes were expressed in B16 mouse melanoma cells and then studied the effect of ATRA, 8-bromo-cyclic AMP (8BrcA), and phorbol dibutyrate (PDB) on RXR mRNA levels. ATRA induces differentiation in these cells while 8BrcA and PDB antagonize the RA-induced differentiation of B16 melanoma cells. Northern analysis demonstrated the expression of RXR alpha and RXR beta mRNA in B16 cells, but RXR gamma was not detectable. Further analysis using RT-PCR also failed to detect RXR gamma in these cells. Long-term RA treatment decreased the expression of RXR alpha, but not RXR beta mRNAs. PDB did not alter the expression of either RXR mRNAs, however, 8BrcA treatment resulted in a time dependent decrease in the amount of RXR beta, but not RXR alpha mRNA. Inhibition of protein synthesis by cycloheximide resulted in a large increase in RXR alpha and RXR beta mRNA levels. This effect of cycloheximide was time and concentration dependent with maximal stimulation of RXR alpha and RXR beta mRNAs occurring at 4 h of treatment. Inhibition of transcription with actinomycin D completely abolished the cycloheximide-induced increase of RXR beta. In contrast to its effect on other genes, such as immediate response genes, cycloheximide treatment did not increase the half-life of RXR beta mRNA. Nuclear run-on assays showed that cycloheximide treatment of intact B16 melanoma cells stimulated the transcription rate of RXR beta, but not RXR alpha. These results suggest the presence of an unstable transcription factor that negatively regulates the expression of RXR beta in B16 melanoma cells. In addition, since RXR beta is the predominant isotype in B16 cells, 8BrcA may, at least partially, inhibit RA-induced differentiation through down-regulation of this RXR.

Correlation of retinoid binding affinity to retinoic acid receptor alpha with retinoid inhibition of growth of estrogen receptor-positive MCF-7 mammary carcinoma cells.

Both anchorage-dependent growth and anchorage-independent growth of the estrogen receptor-positive mammary carcinoma cell line MCF-7 are inhibited by all-trans-retinoic acid. This cell line has nuclear retinoic acid receptors (RARs) alpha and gamma. The natural retinoids all-trans-retinoic acid and 9-cis-retinoic acid and a series of 12 conformationally restricted retinoids, which showed a range of binding selectivities for these receptors and had either agonist or antagonist activity for gene transcriptional activation by the RARs, were evaluated for their abilities to inhibit anchorage-dependent (adherent) and anchorage-independent (clonal) growth of MCF-7 cells. Correlation analyses were performed to relate growth inhibition by these retinoids with their binding affinity to RAR alpha or RAR gamma. Inhibition of anchorage-dependent growth in culture after 7 days of retinoid treatment correlated with binding to RAR alpha (n = 14; P < or = 0.001) and not to RAR gamma (n = 14; P > 0.1). Both the RAR alpha-selective retinoid agonists and the two RAR antagonists that were evaluated inhibited adherent cell growth. The RAR gamma-selective agonists had very low growth inhibitory activity (< 10%) at concentrations as high as 12.5 microM. These results suggest that RAR alpha is the retinoid receptor involved in the inhibition of adherent cell growth by retinoids and that transcriptional activation by this receptor on a RAR response element does not appear to be required for this process to occur. For this series of retinoids, inhibition of anchorage-independent growth after 21 days of retinoid treatment only correlated (n = 12; P < or = 0.005) with binding affinity to RAR alpha for the retinoid agonists, although the RAR gamma-selective retinoids displayed weak activity. The RAR antagonists were very poor inhibitors of growth. These results suggest that activation of gene transcription by RAR alpha appears to be required for inhibition of anchorage-independent growth by retinoids in this estrogen receptor-positive mammary carcinoma cell line.

Use of vitamins A and D in chemoprevention and therapy of cancer: control of nuclear receptor expression and function. Vitamins, cancer and receptors.

Vitamin A is metabolized to several biologically active compounds, the best known of which is retinoic acid. This compound has been shown to inhibit the growth of a variety of tumor cells and to induce a more differentiated phenotype in several tumor types. Vitamin D is metabolized to the active compound 1,25-dihydroxyvitamin D3. This vitamin is well-known for its role in maintaining calcium homeostasis in the body. Recently it has been shown that vitamin D3 can also inhibit tumor cell replication and stimulate differentiation of selected tumor types. Retinoic acid is being used clinically to treat promyelocytic leukemia, head and neck tumors as well as cervical dysplasia. Use of vitamin D3 clinically has been restricted by its affect on calcium metabolism. Recently, however, new analogs of vitamin D3 have been developed which have much less calcium mobilizing activity, yet still retain their tumor inhibitory properties. The action of both of these vitamins is mediated by nuclear receptors which have the same structure as steroid receptors. There are three nuclear retinoic acid receptors (RAR alpha, beta, and gamma), but only one vitamin D3 nuclear receptor. These receptors are expressed in very small amounts. Since the ligand should be in vast excess of receptor (ie not limiting), we explored the possibility that response to vitamin A might be mediated by control of RAR expression. Using B16 mouse melanoma cells as a model system, we found that RAR alpha and gamma mRNAs were constitutively expressed. RAR beta mRNA was induced by treatment of the cells with RA. Induction of RAR beta mRNA occurred within 1h and was not inhibited by cycloheximide. The mRNA for all three RARs was dramatically decreased with 8-bromo-cyclic AMP treatment and could not be rescued by addition of RA. Analysis of RAR gamma revealed that this decrease occurred within 1h of exposure to 8-bromo-cyclic AMP and was not blocked by simultaneous treatment with cycloheximide. Nuclear extracts from cyclic AMP-treated cells showed a large decrease in protein binding to a retinoic acid response element (RARE) oligonucleotide compared to control cells. This correlated with a marked reduction of RA-stimulated RARE-reporter gene activity in transfected cells which were treated with cyclic AMP. Pre-treatment of B16 cells with cyclic AMP prior to RA addition dramatically reduced induction of PKC alpha, an early marker of RA-induced cell differentiation. Thus, cyclic AMP can antagonize the physiological actions of RA via its ability to inhibit RAR expression.

Characterization of conventional protein kinase C (PKC) isotype expression during F9 teratocarcinoma differentiation. Overexpression of PKC alpha alters the expression of some differentiation-dependent genes.

F9 teratocarcinoma is a useful model for studying early embryogenesis since these cells can differentiate into primitive or parietal endoderm under the influence of retinoic acid or retinoic acid and cyclic AMP, respectively. We have found that three isoforms of protein kinase C (PKC alpha, -beta, and -gamma) were expressed in undifferentiated stem cells. When the cells were treated with retinoic acid either alone or in the presence of cAMP for 120 h, PKC alpha mRNA and protein levels increased, whereas those of PKC beta and PKC gamma became undetectable. These changes began within 24 h of drug treatment and were complete by 48-72 h. In order to determine the functional significance of the induction of PKC alpha during F9 differentiation, we established two stable transfectants that overexpressed PKC alpha protein between 4- and 5-fold compared to wild type cells. Characterization of these cell lines revealed an altered pattern of expression of some of the markers of F9 differentiation. The clone that had the highest amount of PKC alpha protein constitutively expressed mRNA for type IV collagen and c-Jun, which are not normally expressed until 24-48 h of treatment with differentiation agents. In the other overexpressing clone, these markers were induced much faster than in wild type cells. The growth rate of both overexpressing clones was less than wild type cells, while the expression of the PKC beta protein in these clones was similar to the levels found in differentiated F9 cells. However, other markers of differentiation, including the cellular morphology and levels of pST6-135 and c-myc RNA, responded to agents identically in both wild type and PKC-alpha-overexpressing clones. Therefore, overexpression of PKC alpha is not sufficient to induce full differentiation of F9 cells. However, our data suggest that certain pathways that lead to the expression of differentiation-dependent genes are regulated by PKC alpha protein levels.

Expression of TGF-beta during in vitro differentiation of hamster tracheal epithelial cells.

The control of growth and differentiation of tracheal epithelial cells is poorly understood. Retinoic acid seems to be essential for the growth and secretory cell differentiation of hamster tracheal epithelial (HTE) cells in culture. In this study, we tested the hypothesis that one way by which retinoic acid (RA) stimulates growth is by decreasing transforming growth factor beta (TGF beta) expression or activity or both. HTE cells were very sensitive to TGF beta-induced growth inhibition. TGF beta 1 was more potent than TGF beta 2 with 50% inhibition of growth achieved at a concentration less than 0.1 ng/ml. A single TGF beta 1 transcript of 2.4 kb was expressed in HTE cells, and the amount increased by fourfold as cell proliferation decreased and differentiation increased. No TGF beta 2 mRNA could be detected in proliferating undifferentiated HTE cells, but two distinct mRNAs (5.1 and 3.5 kb) were observed to be induced in a transient fashion in RA-treated cells which correlated with the onset of differentiation. The amount of biologically active TGF beta in conditioned media from HTE cells at different stages of growth and differentiation in primary culture was determined by the mink lung epithelial cell growth inhibition assay and the use of neutralizing antibodies. These assays indicated a large increase in the total amount of TGF beta at the time the cells slowed their growth and started to differentiate. The activity was due primarily to TGF beta 1. Interestingly, cells treated with RA had a major component of "preactivated" (non-latent) TGF beta 1 compared to control cells.(ABSTRACT TRUNCATED AT 250 WORDS)